Patent application title: GENE SMS 27

Abstract:

The present invention relates to newly identified genes that encode
proteins that are involved in the synthesis of L-ascorbic acid
(hereinafter also referred to as Vitamin C). The invention also features
polynucleotides comprising the full-length polynucleotide sequences of
the novel genes and fragments thereof, the novel polypeptides encoded by
the polynucleotides and fragments thereof, as well as their functional
equivalents. The present invention also relates to the use of said
polynucleotides and polypeptides as biotechnological tools in the
production of Vitamin C from microorganisms, whereby a modification of
said polynucleotides and/or encoded polypeptides has a direct or indirect
impact on yield, production, and/or efficiency of production of the
fermentation product in said microorganism. Also included are
methods/processes of using the polynucleotides and modified
polynucleotide sequences to transform host microorganisms. The invention
also relates to genetically engineered microorganisms and their use for
the direct production of Vitamin C.

Claims:

1. A polynucleotide selected from the group consisting of:(a)
polynucleotides encoding a polypeptide comprising the amino acid sequence
according to SEQ ID NO:2;(b) polynucleotides comprising the nucleotide
sequence according to SEQ ID NO:1;(c) polynucleotides comprising a
nucleotide sequence obtainable by nucleic acid amplification such as
polymerase chain reaction, using genomic DNA from a microorganism as a
template and a primer set according to SEQ ID NO:3 and SEQ ID NO:4;(d)
polynucleotides comprising a nucleotide sequence encoding a fragment or
derivative of a polypeptide encoded by a polynucleotide of any of (a) to
(c) wherein in said derivative one or more amino acid residues are
conservatively substituted compared to said polypeptide, and said
fragment or derivative has the activity of a transcriptional regulator,
preferably a repressor of L-sorbose dehydrogenase (SDH) and/or
L-sorbosone dehydrogenase (SNDH);(e) polynucleotides the complementary
strand of which hybridizes under stringent conditions to a polynucleotide
as defined in any one of (a) to (d) and which encode a transcriptional
regulator, preferably a repressor of L-sorbose dehydrogenase (SDH) and/or
L-sorbosone dehydrogenase (SNDH); and(f) polynucleotides which are at
least 60%, such as 70, 85, 90 or 95% identical to a polynucleotide as
defined in any one of (a) to (d) and which encode a transcriptional
regulator, preferably a repressor of L-sorbose dehydrogenase (SDH) and/or
L-sorbosone dehydrogenase (SNDH);or the complementary strand of such a
polynucleotide.

2. A vector containing the polynucleotide according to claim 1.

3. The vector of claim 2 in which the polynucleotide is operatively linked
to expression control sequences allowing the expression in prokaryotic or
eukaryotic host cells.

4. A microorganism genetically engineered with a polynucleotide according
to claim 1.

5. A microorganism according to claim 4 capable of directly producing
Vitamin C from D-sorbitol in quantities of 300 mg/l or more when measured
in a resting cell method after an incubation period of 20 hours.

6. A microorganism according to claim 5 capable of directly producing
Vitamin C from L-sorbose in quantities of 800 mg/l or more.

7. A polypeptide encoded by a polynucleotide according to claim 1.

8. Process for producing cells capable of expressing a polypeptide
according to claim 7, comprising the step of genetically engineering
cells with the vector.

9. Use of a disrupted polynucleotide according to claim 1 for the
production of Vitamin C and/or 2-KGA.

10. A microorganism according to claim 4 or a microorganism containing an
endogenous gene comprising a polynucleotide, said microorganism being
genetically altered in such a way that it leads to an improved yield
and/or efficiency of production of Vitamin C and/or 2-KGA produced by
said microorganism.

11. A microorganism according to claim 10 producing a polypeptide with
decreased or abolished activity of a transcriptional regulator,
preferably a repressor of L-sorbose dehydrogenase (SDH) and/or
L-sorbosone dehydrogenase (SNDH) gene.

12. A microorganism according to claim 4 wherein the polynucleotide is
disrupted.

14. Process for the production of an disrupted endogenous transcriptional
regulator gene, preferably a repressor of L-sorbose dehydrogenase (SDH)
and/or L-sorbosone dehydrogenase (SNDH) gene in a microorganism, said
microorganism comprising a polynucleotide according to claim 1, said
process comprising the step of altering said polynucleotide in such a way
that it leads to an improved yield and/or efficiency of production of
Vitamin C and/or 2-KGA produced by said microorganism.

15. Process for the production of a microorganism capable of producing
Vitamin C and/or 2-KGA, comprising the step of altering said
microorganism so that the microorganism produces a polypeptide with
reduced or abolished activity of a transcriptional regulator, preferably
a repressor of L-sorbose dehydrogenase (SDH) and/or L-sorbosone
dehydrogenase (SNDH) gene leading to an improved yield and/or efficiency
of production of Vitamin C and/or 2-KGA produced by said microorganism.

16. Process for the production of a microorganism containing an endogenous
gene comprising a polynucleotide according to claim 1, comprising the
step of altering said microorganism so that the endogenous gene is
underexpressed or disrupted, leading to an improved yield and/or
efficiency of production of Vitamin C and/or 2-KGA produced by said
microorganism.

17. Process according to claim 15 the production of a microorganism.

18. Microorganism obtainable by a process according to claim 14.

19. Process for the production of Vitamin C and/or 2-KGA with a
microorganism according to claim 10 wherein said microorganism is
incubated/cultivated in a aqueous medium under conditions that allow the
direct production of Vitamin C and/or 2-KGA from D-sorbitol or L-sorbose
and wherein optionally Vitamin C and/or 2-KGA is isolated as the
fermentation product.

Description:

[0001]The present invention relates to newly identified genes that encode
proteins that are involved in the synthesis of L-ascorbic acid
(hereinafter also referred to as Vitamin C) and/or 2-keto-L-gulonic acid
(hereinafter also referred to as 2-KGA). The invention also features
polynucleotides comprising the full-length polynucleotide sequences of
the novel genes and fragments thereof, the novel polypeptides encoded by
the polynucleotides and fragments thereof, as well as their functional
equivalents. The present invention also relates to the use of said
polynucleotides and polypeptides as biotechnological tools in the
production of Vitamin C and/or 2-KGA from microorganisms, whereby a
modification of said polynucleotides and/or encoded polypeptides has a
direct or indirect impact on yield, production, and/or efficiency of
production of the fermentation product in said microorganism. Also
included are methods/processes of using the polynucleotides and modified
polynucleotide sequences to transform host microorganisms. The invention
also relates to genetically engineered microorganisms and their use for
the direct production of Vitamin C and/or 2-KGA.

[0002]Vitamin C is one of very important and indispensable nutrient
factors for human beings. Vitamin C is also used in animal feed even
though some farm animals can synthesize it in their own body.

[0003]For the past 70 years, Vitamin C has been produced industrially from
D-glucose by the well-known Reichstein method. All steps in this process
are chemical except for one (the conversion of D-sorbitol to L-sorbose),
which is carried out by microbial conversion. Since its initial
implementation for industrial production of Vitamin C, several chemical
and technical modifications have been used to improve the efficiency of
the Reichstein method. Recent developments of Vitamin C production are
summarized in Ullmann's Encyclopedia of Industrial Chemistry, 5th
Edition, Vol. A27 (1996), pp. 547ff.

[0004]Different intermediate steps of Vitamin C production have been
performed with the help of microorganisms or enzymes isolated therefrom.
Thus, 2-keto-L-gulonic acid (2-KGA), an intermediate compound that can be
chemically converted into Vitamin C by means of an alkaline rearrangement
reaction, may be produced by a fermentation process starting from
L-sorbose or D-sorbitol, by means of strains belonging e.g. to the
Ketogulonicigenium or Gluconobacter genera, or by an alternative
fermentation process starting from D-glucose, by means of recombinant
strains belonging to the Gluconobacter or Pantoea genera.

[0005]Current chemical production methods for Vitamin C have some
undesirable characteristics such as high-energy consumption and use of
large quantities of organic and inorganic solvents. Therefore, over the
past decades, other approaches to manufacture Vitamin C using microbial
conversions, which would be more economical as well as ecological, have
been investigated.

[0006]Direct Vitamin C production from a number of substrates including
D-sorbitol, L-sorbose and L-sorbosone has been reported in several
microorganisms, such as algae, yeast and acetic acid bacteria, using
different cultivation methods. Examples of known bacteria able to
directly produce Vitamin C include, for instance, strains from the genera
of Gluconobacter, Gluconacetobacter, Acetobacter, Ketogulonicigenium,
Pantoea, Pseudomonas or Escherichia. Examples of known yeast or algae
include, e.g., Candida, Saccharomyces, Zygosaccharomyces,
Schizosaccharomyces, Kluyveromyces or Chlorella.

[0007]Microorganisms able to assimilate D-sorbitol for growth usually
possess enzymes able to oxidize this compound into a universal
assimilation substrate such as D-fructose. Also microorganisms able to
grow on L-sorbose possess an enzyme, NAD(P)H-dependent L-sorbose
reductase, which is able to reduce this compound to D-sorbitol, which is
then further oxidized into D-fructose. D-fructose is an excellent
substrate for the growth of many microorganisms, after it has been
phosphorylated by means of a D-fructose kinase.

[0008]For instance, in the case of acetic acid bacteria, which are
obligate aerobe, gram-negative microorganisms belonging to the genus
Acetobacter, Gluconobacter, and Gluconacetobacter, these microorganisms
are able to transport D-sorbitol into the cytosol and convert it into
D-fructose by means of a cytosolic NAD-dependent D-sorbitol
dehydrogenase. Some individual strains, such as Gluconobacter oxydans IFO
3292, and IFO 3293, are able as well to transport L-sorbose into the
cytosol and reduce it to D-sorbitol by means of a cytosolic
NAD(P)H-dependent L-sorbose reductase, which then is further oxidized
into D-fructose. In these bacteria, the Embden-Meyerhof-Parnas pathway,
as well as the tricarboxyclic acid cycle are not fully active, and the
main pathway channeling sugars into the central metabolism is the pentose
phosphate pathway. D-fructose-6-phosphate, obtained from D-fructose by a
phosphorylation reaction enters the pentose phosphate pathway, being
further metabolized and producing reducing power in the form of NAD(P)H
and tricarboxylic compounds necessary for growth and maintenance.

[0009]Acetic acid bacteria are well known for their ability to
incompletely oxidize different substrates such as alcohols, sugars, sugar
alcohols and aldehydes. These processes are generally known as oxidative
fermentations or incomplete oxidations, and they have been well
established for a long time in the food and chemical industry, especially
in vinegar and in L-sorbose production. A useful product known to be
obtained from incomplete oxidations of D-sorbitol or L-sorbose using
strains belonging to the Gluconobacter genus is 2-KGA.

[0010]Acetic acid bacteria accomplish these incomplete oxidation reactions
by means of different dehydrogenases located either in the periplasmic
space, on the periplasmic membrane as well as in the cytoplasm. Different
co-factors are employed by the different dehydrogenases, the most common
being PQQ and FAD for membrane-bound or periplasmic enzymes, and NAD/NADP
for cytoplasmic enzymes.

[0011]While all products of these oxidation reactions diffuse back to the
external aqueous environment through the outer membrane, some of them can
be passively or actively transported into the cell and be further used in
metabolic pathways responsible for growth and energy formation. Inside
the cell, oxidized products can many times be reduced back to their
original substrate by means of reductases, and then be channeled back to
the central metabolism.

[0012]Proteins, in particular enzymes and transporters, that are active in
the metabolization of D-sorbitol or L-sorbose are herein referred to as
being involved in the Sorbitol/Sorbose Metabolization System. Such
proteins are abbreviated herein as SMS proteins and function in the
direct metabolization of D-sorbitol or L-sorbose. Metabolization of
D-sorbitol or L-sorbose includes on one side the assimilation of these
compounds into the cytosol and further conversion into metabolites useful
for assimilation pathways such as the Embden-Meyerhof-Parnas pathway, the
pentose phosphate pathway, the Entner-Doudoroff pathway, and the
tricarboxyclic acid cycle, all of them involved in all vital
energy-forming and anabolic reactions necessary for growth and
maintenance of living cells. On the other side, metabolization of
D-sorbitol or L-sorbose also includes the conversion of these compounds
into further oxidized products such as L-sorbosone, 2-KGA and Vitamin C
by so-called incomplete oxidation processes.

[0013]An object of the present invention is to improve the yields and/or
productivity of Vitamin C and/or 2-KGA production.

[0014]Surprisingly, it has now been found that SMS proteins or subunits of
such proteins having activity towards or which are involved in the
assimilation or conversion of D-sorbitol, L-sorbose or L-sorbosone play
an important role in the biotechnological production of Vitamin C and/or
2-KGA.

[0015]In one embodiment, SMS proteins of the present invention are
selected from oxidoreductases [EC 1], preferably oxidoreductases acting
on the CH--OH group of donors [EC 1.1], more preferably oxidoreductases
with NAD.sup.+ or NADP.sup.+ as acceptor [EC 1.1.1] and oxidoreductases
with other acceptors [EC 1.1.99], most preferably selected from
oxidoreductases belonging to enzyme classes [EC 1.1.1.1], [EC 1.1.1.15]
or [EC 1.2.1.-], or preferably oxidoreductases acting on the aldehyde or
oxo group of donors [EC 1.2], more preferably oxidoreductases with
NAD.sup.+ or NADP.sup.+ as acceptor [EC 1.2.1].

[0017]In particular, it has now been found that SMS proteins encoded by
polynucleotides having a nucleotide sequence that hybridizes preferably
under highly stringent conditions to a sequence shown in SEQ ID NO:1 play
an important role in the biotechnological production of Vitamin C and/or
2-KGA. It has also been found, that by genetically altering the
expression level of nucleotides according to the invention in a
microorganism capable of directly producing Vitamin C, such as for
example Gluconobacter, the direct fermentation of Vitamin C and/or 2-KGA
by said microorganism can be even greatly improved.

[0018]Consequently, the invention relates to a polynucleotide selected
from the group consisting of:

(a) polynucleotides encoding a polypeptide comprising the amino acid
sequence according to SEQ ID NO:2;(b) polynucleotides comprising the
nucleotide sequence according to SEQ ID NO:1;(c) polynucleotides
comprising a nucleotide sequence obtainable by nucleic acid amplification
such as polymerase chain reaction, using genomic DNA from a microorganism
as a template and a primer set according to SEQ ID NO:3 and SEQ ID
NO:4;(d) polynucleotides comprising a nucleotide sequence encoding a
fragment or derivative of a polypeptide encoded by a polynucleotide of
any of (a) to (c) wherein in said derivative one or more amino acid
residues are conservatively substituted compared to said polypeptide, and
said fragment or derivative has the activity of a transcriptional
regulator, preferably a repressor of L-sorbose dehydrogenase (SDH) and/or
L-sorbosone dehydrogenase (SNDH) (SMS 27);(e) polynucleotides the
complementary strand of which hybridizes under stringent conditions to a
polynucleotide as defined in any one of (a) to (d) and which encode a
transcriptional regulator, preferably a repressor of L-sorbose
dehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH) (SMS 27);
and(f) polynucleotides which are at least 60%, such as 70, 85, 90 or 95%
identical to a polynucleotide as defined in any one of (a) to (d) and
which encode a transcriptional regulator, preferably a repressor of
L-sorbose dehydrogenase (SDH) and/or L-sorbosone dehydrogenase (SNDH)
(SMS 27); orthe complementary strand of such a polynucleotide.

[0019]The SMS protein as isolated from Gluconobacter oxydans IFO 3293
shown in SEQ ID NO:2 and described herein was found to be a particularly
useful SMS protein, since it appeared that it performs a crucial function
in the direct Vitamin C production in microorganisms, in particular in
bacteria, such as acetic acid bacteria, such as Gluconobacter,
Acetobacter and Gluconacetobacter. Accordingly, the invention relates to
a polynucleotide encoding a polypeptide according to SEQ ID NO:2. This
protein may be encoded by a nucleotide sequence as shown in SEQ ID NO:1.
The invention therefore also relates to polynucleotides comprising the
nucleotide sequence according to SEQ ID NO:1.

[0020]The nucleotide and amino acid sequences determined above were used
as a "query sequence" to perform a search with Blast2 program (version 2
or BLAST from National Center for Biotechnology [NCBI] against the
database PRO SW-SwissProt (full release plus incremental updates). From
the searches, the SMS 27 polynucleotide according to SEQ ID NO:1 was
annotated as encoding a transcriptional regulator belonging to the
TetR/AcrR-family. The protein as encoded by SEQ ID NO:2 acts as a
repressor by directly binding to the promoter region of the respective
genes, including the genes coding for L-sorbose dehydrogenase (SDH), such
as e.g. shown in SEQ ID NO:11 encoding a protein as of SEQ ID NO:12,
L-sorbosone dehydrogenase (SNDH), such as e.g. shown in SEQ ID NO:13
encoding a protein as of SEQ ID NO:14, and L-sorbosone exporter, such as
e.g. shown in SEQ ID NO:15 encoding a protein as of SEQ ID NO:16.

[0021]A nucleic acid according to the invention may be obtained by nucleic
acid amplification using cDNA, mRNA or alternatively, genomic DNA, as a
template and appropriate oligonucleotide primers such as the nucleotide
primers according to SEQ ID NO:3 and SEQ ID NO:4 according to standard
PCR amplification techniques. The nucleic acid thus amplified may be
cloned into an appropriate vector and characterized by DNA sequence
analysis.

[0022]The template for the reaction may be cDNA obtained by reverse
transcription of mRNA prepared from strains known or suspected to
comprise a polynucleotide according to the invention. The PCR product may
be subcloned and sequenced to ensure that the amplified sequences
represent the sequences of a new nucleic acid sequence as described
herein, or a functional equivalent thereof.

[0023]The PCR fragment may then be used to isolate a full length cDNA
clone by a variety of known methods. For example, the amplified fragment
may be labeled and used to screen a bacteriophage or cosmid cDNA library.
Alternatively, the labeled fragment may be used to screen a genomic
library.

[0024]Accordingly, the invention relates to polynucleotides comprising a
nucleotide sequence obtainable by nucleic acid amplification such as
polymerase chain reaction, using DNA such as genomic DNA from a
microorganism as a template and a primer set according to SEQ ID NO:3 and
SEQ ID NO:4.

[0025]The invention also relates to polynucleotides comprising a
nucleotide sequence encoding a fragment or derivative of a polypeptide
encoded by a polynucleotide as described herein wherein in said
derivative one or more amino acid residues are conservatively substituted
compared to said polypeptide, and said fragment or derivative has the
activity of a SMS polypeptide, preferably a SMS 27 polypeptide.

[0026]The invention also relates to polynucleotides the complementary
strand of which hybridizes under stringent conditions to a polynucleotide
as defined herein and which encode a SMS polypeptide, preferably a SMS 27
polypeptide.

[0027]The invention also relates to polynucleotides which are at least 60%
identical to a polynucleotide as defined herein and which encode a SMS
polypeptide; and the invention also relates to polynucleotides being the
complementary strand of a polynucleotide as defined herein above.

[0028]The invention also relates to primers, probes and fragments that may
be used to amplify or detect a DNA according to the invention and to
identify related species or families of microorganisms also carrying such
genes.

[0029]The present invention also relates to vectors which include
polynucleotides of the invention and microorganisms which are genetically
engineered with the polynucleotides or said vectors.

[0030]The invention also relates to processes for producing microorganisms
capable of expressing a polypeptide encoded by the above defined
polynucleotide and a polypeptide encoded by a polynucleotide as defined
above.

[0031]The invention also relates to microorganisms wherein the activity of
a SMS polypeptide, preferably a SMS 27 polypeptide, is reduced or
abolished so that the yield of Vitamin C and/or 2-KGA which is directly
produced from D-sorbitol or L-sorbose is increased.

[0032]The skilled person will know how to reduce or abolish the activity
of a SMS protein, preferably a SMS 27 protein. Such may be for instance
accomplished by either genetically modifying the host organism in such a
way that it produces less or no copies of the SMS protein, preferably the
SMS 27 protein, than the wild type organism or by decreasing or
abolishing the specific activity of the SMS protein, preferably the SMS
27 protein.

[0033]In the following description, procedures are detailed to achieve
this goal, i.e. the increase in the yield and/or production of Vitamin C
which is directly produced from D-sorbitol or L-sorbose by reducing or
abolishing the activity of a SMS 27 protein. These procedures apply
mutatis mutandis for other SMS proteins.

[0034]Modifications in order to have the organism produce less or no
copies of the SMS 27 gene and/or protein may include the use of a weak
promoter, or the mutation (e.g. insertion, deletion or point mutation) of
(parts of) the SMS 27 gene or its regulatory elements. Decreasing or
abolishing the specific activity of a SMS 27 protein may also be
accomplished by methods known in the art. Such methods may include the
mutation (e.g. insertion, deletion or point mutation) of (parts of) the
SMS 27 gene. This may for instance affect the interaction with DNA that
is mediated by the N-terminal region of SMS 27 or interaction with other
effector molecules.

[0035]Also known in the art are methods of reducing or abolishing the
activity of a given protein by contacting the SMS 27 protein with
specific inhibitors or other substances that specifically interact with
the SMS 27 protein. In order to identify such specific inhibitors, the
SMS 27 protein may be expressed and tested for activity in the presence
of compounds suspected to inhibit the activity of the SMS 27 protein.
Potential inhibiting compounds may for instance be monoclonal or
polyclonal antibodies against the SMS 27 protein. Such antibodies may be
obtained by routine immunization protocols of suitable laboratory
animals.

[0036]The invention may be performed in or with any microorganism carrying
a SMS 27 gene or equivalent or homologue thereof. Suitable microorganisms
may be selected from bacteria, either as wild type strains, mutant
strains derived by classic mutagenesis and selection methods or as
recombinant strains. Examples of such bacteria may be, e.g.,
Gluconobacter, Gluconacetobacter, Acetobacter, Ketogulonicigenium,
Pantoea, Rhizobium, Sinohrizobium, such as Sinorhizobium meliloti,
Bradyrhizobium, such as Bradyrhizobium japonicum, Roseobacter, Ralstonia,
Pseudomonas, such as, e.g., Pseudomonas putida, and Escherichia, such as,
e.g., Escherichia coli. Preferred are Gluconobacter or Acetobacter aceti,
such as for instance G. oxydans, G. cerinus, G. frateurii, A. aceti
subsp. xylinum or A. aceti subsp. orleanus, preferably G. oxydans IFO
3293, and their derivatives carrying genes involved in Vitamin C
production pathways and the adjacent regions where the SMS 27 gene or its
equivalent might be located.

[0038]A microorganism as of the present invention may carry further
modifications either on the DNA or protein level (see above), as long as
such modification has a direct impact on the yield, production and/or
efficiency of the direct production of Vitamin C and/or 2-KGA from
substrates like e.g. D-sorbitol or L-sorbose. Such further modifications
may for instance affect other genes encoding SMS proteins as described
above, in particular genes encoding membrane-bound L-sorbosone
dehydrogenases, such as L-sorbosone dehydrogenase SNDHai, membrane-bound
PQQ bound D-sorbitol dehydrogenases and/or other genes encoding proteins
involved transport of sugar and/or sugar alcohols, such as e.g.
exporters, in particular sorbosone exporters, preferably a gene as shown
in SEQ ID NO:15. Methods of performing such modifications are known in
the art, with some examples further described herein. For the use of
SNDHai for direct production of Vitamin C as well as the nucleotide and
amino acid sequence thereof we refer to WO 2005/017159 which is
incorporated herein by reference.

[0039]In accordance with a further object of the present invention there
is provided the use of a polynucleotide as defined above or a
microorganism which is genetically engineered using such polynucleotides
in the production of Vitamin C and/or 2-KGA.

[0040]The invention also relates to processes for the expression of
endogenous genes in a microorganism, to processes for the production of
polypeptides as defined above in a microorganism and to processes for the
production of microorganisms capable of producing Vitamin C and/or 2-KGA.
All these processes may comprise the step of altering a microorganism,
wherein "altering" as used herein encompasses the process for
"genetically altering" or "altering the composition of the cell culture
media and/or methods used for culturing" in such a way that the yield
and/or productivity of the fermentation product can be improved compared
to the wild-type organism. As used herein, "improved yield of Vitamin C"
means an increase of at least 5%, 10%, 25%, 30%, 40%, 50%, 75%, 100%,
200% or even more than 500%, compared to a wild-type microorganism, i.e.
a microorganism which is not genetically altered.

[0041]The term "genetically engineered" or "genetically altered" means the
scientific alteration of the structure of genetic material in a living
organism. It involves the production and use of recombinant DNA. More in
particular it is used to delineate the genetically engineered or modified
organism from the naturally occurring organism. Genetic engineering may
be done by a number of techniques known in the art, such as e.g. gene
replacement, gene amplification, gene disruption, transfection,
transformation using plasmids, viruses, or other vectors. A genetically
modified organism, e.g. genetically modified microorganism, is also often
referred to as a recombinant organism, e.g. recombinant microorganism.

[0042]In accordance with still another aspect of the invention there is
provided a process for the production of Vitamin C and/or 2-KGA by direct
fermentation.

[0043]Several substrates may be used as a carbon source in a process of
the present invention, i.e. a process for direct conversion of a given
substrate into Vitamin C such as e.g. mentioned above. Particularly
suited carbon sources are those that are easily obtainable from the
D-glucose or D-sorbitol metabolization pathway such as, for example,
D-glucose, D-sorbitol, L-sorbose, L-sorbosone, 2-keto-L-gluconate,
D-gluconate, 2-keto-D-gluconate or 2,5-diketo-gluconate. Preferably, the
substrate is selected from for instance D-glucose, D-sorbitol, L-sorbose
or L-sorbosone, more preferably from D-glucose, D-sorbitol or L-sorbose,
and most preferably from D-sorbitol, L-sorbose or L-sorbosone. The term
"substrate" and "production substrate" in connection with the above
process using a microorganism is used interchangeably herein.

[0044]A medium as used herein for the above process using a microorganism
may be any suitable medium for the production of Vitamin C and/or 2-KGA.
Typically, the medium is an aqueous medium comprising for instance salts,
substrate(s), and a certain pH. The medium in which the substrate is
converted into Vitamin C and/or 2-KGA is also referred to as the
production medium.

[0045]"Fermentation" or "production" or "fermentation process" as used
herein may be the use of growing cells using media, conditions and
procedures known to the skilled person, or the use of non-growing
so-called resting cells, after they have been cultivated by using media,
conditions and procedures known to the skilled person, under appropriate
conditions for the conversion of suitable substrates into desired
products such as Vitamin C and/or 2-KGA. Preferably, resting cells are
used for the production of Vitamin C. An example of such process for the
production of Vitamin C is described in WO 2005/017159 (as incorporated
herein by reference). Preferably, 2-KGA is produced using growing cells
(see, e.g. EP 0 518 136 B1).

[0046]The term "direct fermentation", "direct production", "direct
conversion" and the like is intended to mean that a microorganism is
capable of the conversion of a certain substrate into the specified
product by means of one or more biological conversion steps, without the
need of any additional chemical conversion step. For instance, the term
"direct conversion of D-sorbitol into Vitamin C" is intended to describe
a process wherein a microorganism is producing Vitamin C and wherein
D-sorbitol is offered as a carbon source without the need of an
intermediate chemical conversion step. A single microorganism capable of
directly fermenting Vitamin C is preferred. Said microorganism is
cultured under conditions which allow such conversion from the substrate
as defined above.

[0047]In connection with the above process using a microorganism it is
understood that the above-mentioned microorganisms also include synonyms
or basonyms of such species having the same physiological properties, as
defined by the International Code of Nomenclature of Prokaryotes. The
nomenclature of the microorganisms as used herein is the one officially
accepted (at the filing date of the priority application) by the
International Committee on Systematics of Prokaryotes and the
Bacteriology and Applied Microbiology Division of the International Union
of Microbiological Societies, and published by its official publication
vehicle International Journal of Systematic and Evolutionary Microbiology
(IJSEM). A particular reference is made to Urbance et al., IJSEM (2001)
vol 51:1059-1070, with a corrective notification on IJSEM (2001) vol
51:1231-1233, describing the taxonomically reclassification of G. oxydans
DSM 4025 as Ketogulonicigenium vulgare.

[0048]As used herein, resting cells refer to cells of a microorganism
which are for instance viable but not actively growing, or which are
growing at low specific growth rates, for instance, growth rates that are
lower than 0.02 h-1, preferably lower than 0.01 h-1. Cells
which show the above growth rates are said to be in a "resting cell
mode".

[0049]The process of the present invention as above using a microorganism
may be performed in different steps or phases: preferably, the
microorganism is cultured in a first step (also referred to as step (a)
or growth phase) under conditions which enable growth. This phase is
terminated by changing of the conditions such that the growth rate of the
microorganism is reduced leading to resting cells, also referred to as
step (b), followed by the production of Vitamin C from the substrate
using the (b), also referred to as production phase.

[0050]Growth and production phase as performed in the above process using
a microorganism may be performed in the same vessel, i.e., only one
vessel, or in two or more different vessels, with an optional cell
separation step between the two phases. The produced Vitamin C can be
recovered from the cells by any suitable means. Recovering means for
instance that the produced Vitamin C may be separated from the production
medium. Optionally, the thus produced Vitamin C may be further processed.

[0051]For the purpose of the present invention relating to the above
process using a microorganism, the terms "growth phase", "growing step",
"growth step" and "growth period" are used interchangeably herein. The
same applies for the terms "production phase", "production step",
"production period".

[0052]One way of performing the above process using a microorganism as of
the present invention may be a process wherein the microorganism is grown
in a first vessel, the so-called growth vessel, as a source for the
resting cells, and at least part of the cells are transferred to a second
vessel, the so-called production vessel. The conditions in the production
vessel may be such that the cells transferred from the growth vessel
become resting cells as defined above. Vitamin C is produced in the
second vessel and recovered therefrom.

[0053]In connection with the above process using a microorganism, in one
aspect, the growing step can be performed in an aqueous medium, i.e. the
growth medium, supplemented with appropriate nutrients for growth under
aerobic conditions. The cultivation may be conducted, for instance, in
batch, fed-batch, semi-continuous or continuous mode. The cultivation
period may vary depending on for instance the host, pH, temperature and
nutrient medium to be used, and may be for instance about 10 h to about
10 days, preferably about 1 to about 10 days, more preferably about 1 to
about 5 days when run in batch or fed-batch mode, depending on the
microorganism. If the cells are grown in continuous mode, the residence
time may be for instance from about 2 to about 100 h, preferably from
about 2 to about 50 h, depending on the microorganism. If the
microorganism is selected from bacteria, the cultivation may be conducted
for instance at a pH of about 3.0 to about 9.0, preferably about 4.0 to
about 9.0, more preferably about 4.0 to about 8.0, even more preferably
about 5.0 to about 8.0. If algae or yeast are used, the cultivation may
be conducted, for instance, at a pH below about 7.0, preferably below
about 6.0, more preferably below about 5.5, and most preferably below
about 5.0. A suitable temperature range for carrying out the cultivation
using bacteria may be for instance from about 13° C. to about
40° C., preferably from about 18° C. to about 37°
C., more preferably from about 13° C. to about 36° C., and
most preferably from about 18° C. to about 33° C. If algae
or yeast are used, a suitable temperature range for carrying out the
cultivation may be for instance from about 15° C. to about
40° C., preferably from about 20° C. to about 45°
C., more preferably from about 25° C. to about 40° C., even
more preferably from about 25° C. to about 38° C., and most
preferably from about 30° C. to about 38° C. The culture
medium for growth usually may contain such nutrients as assimilable
carbon sources, e.g., glycerol, D-mannitol, D-sorbitol, L-sorbose,
erythritol, ribitol, xylitol, arabitol, inositol, dulcitol, D-ribose,
D-fructose, D-glucose, sucrose, and ethanol, preferably L-sorbose,
D-glucose, D-sorbitol, D-mannitol, glycerol and ethanol; and digestible
nitrogen sources such as organic substances, e.g., peptone, yeast extract
and amino acids. The media may be with or without urea and/or corn steep
liquor and/or baker's yeast. Various inorganic substances may also be
used as nitrogen sources, e.g., nitrates and ammonium salts. Furthermore,
the growth medium, usually may contain inorganic salts, e.g., magnesium
sulfate, manganese sulfate, potassium phosphate, and calcium carbonate.
Cells obtained using the procedures described above can then be further
incubated at essentially the same modes, temperature and pH conditions as
described above, in the presence of substrates such as D-sorbitol,
L-sorbose, or D-glucose, in such a way that they convert these substrates
directly into Vitamin C and/or 2-KGA. Incubation can be done in a
nitrogen-rich medium, containing, for example, organic nitrogen sources,
e.g., peptone, yeast extract, baker's yeast, urea, amino acids, and corn
steep liquor, or inorganic nitrogen sources, e.g., nitrates and ammonium
salts, in which case cells will be able to further grow while producing
Vitamin C and/or 2-KGA. Alternatively, incubation can be done in a
nitrogen-poor medium, in which case cells will not grow substantially,
and will be in a resting cell mode, or biotransformation mode. In all
cases, the incubation medium may also contain inorganic salts, e.g.,
magnesium sulfate, manganese sulfate, potassium phosphate, and calcium
chloride.

[0054]In connection with the above process using a microorganism, in the
growth phase the specific growth rates are for instance at least 0.02
h-1. For cells growing in batch, fed-batch or semi-continuous mode,
the growth rate depends on for instance the composition of the growth
medium, pH, temperature, and the like. In general, the growth rates may
be for instance in a range from about 0.05 to about 0.2 h-1,
preferably from about 0.06 to about 0.15 h-1, and most preferably
from about 0.07 to about 0.13 h-1.

[0055]In another aspect of the above process using a microorganism,
resting cells may be provided by cultivation of the respective
microorganism on agar plates thus serving as growth vessel, using
essentially the same conditions, e.g., cultivation period, pH,
temperature, nutrient medium as described above, with the addition of
agar agar.

[0056]In connection with the above process using a microorganism, if the
growth and production phase are performed in two separate vessels, then
the cells from the growth phase may be harvested or concentrated and
transferred to a second vessel, the so-called production vessel. This
vessel may contain an aqueous medium supplemented with any applicable
production substrate that can be converted to Vitamin C by the cells.
Cells from the growth vessel can be harvested or concentrated by any
suitable operation, such as for instance centrifugation, membrane
crossflow ultrafiltration or microfiltration, filtration, decantation,
flocculation. The cells thus obtained may also be transferred to the
production vessel in the form of the original broth from the growth
vessel, without being harvested, concentrated or washed, i.e. in the form
of a cell suspension. In a preferred embodiment, the cells are
transferred from the growth vessel to the production vessel in the form
of a cell suspension without any washing or isolating step in-between.

[0057]Thus, in a preferred embodiment of the above process using a
microorganism step (a) and (c) of the process of the present invention as
described above are not separated by any washing and/or separation step.

[0058]In connection with the above process using a microorganism, if the
growth and production phase are performed in the same vessel, cells may
be grown under appropriate conditions to the desired cell density
followed by a replacement of the growth medium with the production medium
containing the production substrate. Such replacement may be, for
instance, the feeding of production medium to the vessel at the same time
and rate as the withdrawal or harvesting of supernatant from the vessel.
To keep the resting cells in the vessel, operations for cell recycling or
retention may be used, such as for instance cell recycling steps. Such
recycling steps, for instance, include but are not limited to methods
using centrifuges, filters, membrane crossflow microfiltration of
ultrafiltration steps, membrane reactors, flocculation, or cell
immobilization in appropriate porous, non-porous or polymeric matrixes.
After a transition phase, the vessel is brought to process conditions
under which the cells are in a resting cell mode as defined above, and
the production substrate is efficiently converted into Vitamin C.

[0059]The aqueous medium in the production vessel as used for the
production step in connection with the above process using a
microorganism, hereinafter called production medium, may contain only the
production substrate(s) to be converted into Vitamin C, or may contain
for instance additional inorganic salts, e.g., sodium chloride, calcium
chloride, magnesium sulfate, manganese sulfate, potassium phosphate,
calcium phosphate, and calcium carbonate. The production medium may also
contain digestible nitrogen sources such as for instance organic
substances, e.g., peptone, yeast extract, urea, amino acids, and corn
steep liquor, and inorganic substances, e.g. ammonia, ammonium sulfate,
and sodium nitrate, at such concentrations that the cells are kept in a
resting cell mode as defined above. The medium may be with or without
urea and/or corn steep liquor and/or baker's yeast. The production step
may be conducted for instance in batch, fed-batch, semi-continuous or
continuous mode. In case of fed-batch, semi-continuous or continuous
mode, both cells from the growth vessel and production medium can be fed
continuously or intermittently to the production vessel at appropriate
feed rates. Alternatively, only production medium may be fed continuously
or intermittently to the production vessel, while the cells coming from
the growth vessel are transferred at once to the production vessel. The
cells coming from the growth vessel may be used as a cell suspension
within the production vessel or may be used as for instance flocculated
or immobilized cells in any solid phase such as porous or polymeric
matrixes. The production period, defined as the period elapsed between
the entrance of the substrate into the production vessel and the harvest
of the supernatant containing Vitamin C, the so-called harvest stream,
can vary depending for instance on the kind and concentration of cells,
pH, temperature and nutrient medium to be used, and is preferably about 2
to about 100 h. The pH and temperature can be different from the pH and
temperature of the growth step, but is essentially the same as for the
growth step.

[0060]In a preferred embodiment of the above process using a
microorganism, the production step is conducted in continuous mode,
meaning that a first feed stream containing the cells from the growth
vessel and a second feed stream containing the substrate is fed
continuously or intermittently to the production vessel. The first stream
may either contain only the cells isolated/separated from the growth
medium or a cell suspension, coming directly from the growth step, i.e.
cells suspended in growth medium, without any intermediate step of cell
separation, washing and/or isolating. The second feed stream as herein
defined may include all other feed streams necessary for the operation of
the production step, e.g. the production medium comprising the substrate
in the form of one or several different streams, water for dilution, and
base for pH control.

[0061]In connection with the above process using a microorganism, when
both streams are fed continuously, the ratio of the feed rate of the
first stream to feed rate of the second stream may vary between about
0.01 and about 10, preferably between about 0.01 and about 5, most
preferably between about 0.02 and about 2. This ratio is dependent on the
concentration of cells and substrate in the first and second stream,
respectively.

[0062]Another way of performing the process as above using a microorganism
of the present invention may be a process using a certain cell density of
resting cells in the production vessel. The cell density is measured as
absorbance units (optical density) at 600 nm by methods known to the
skilled person. In a preferred embodiment, the cell density in the
production step is at least about 10, more preferably between about 10
and about 200, even more preferably between about 15 and about 200, even
more preferably between about 15 to about 120, and most preferably
between about 20 and about 120.

[0063]In connection with the above process using a microorganism, in order
to keep the cells in the production vessel at the desired cell density
during the production phase as performed, for instance, in continuous or
semi-continuous mode, any means known in the art may be used, such as for
instance cell recycling by centrifugation, filtration, membrane crossflow
ultrafiltration of microfiltration, decantation, flocculation, cell
retention in the vessel by membrane devices or cell immobilization.
Further, in case the production step is performed in continuous or
semi-continuous mode and cells are continuously or intermittently fed
from the growth vessel, the cell density in the production vessel may be
kept at a constant level by, for instance, harvesting an amount of cells
from the production vessel corresponding to the amount of cells being fed
from the growth vessel.

[0064]In connection with the above process using a microorganism, the
produced Vitamin C contained in the so-called harvest stream is
recovered/harvested from the production vessel. The harvest stream may
include, for instance, cell-free or cell-containing aqueous solution
coming from the production vessel, which contains Vitamin C as a result
of the conversion of production substrate by the resting cells in the
production vessel. Cells still present in the harvest stream may be
separated from the Vitamin C by any operations known in the art, such as
for instance filtration, centrifugation, decantation, membrane crossflow
ultrafiltration or microfiltration, tangential flow ultrafiltration or
microfiltration or dead end filtration. After this cell separation
operation, the harvest stream is essentially free of cells.

[0065]In a further aspect, the process of the present invention may be
combined with further steps of separation and/or purification of the
produced Vitamin C from other components contained in the harvest stream,
i.e., so-called downstream processing steps. These steps may include any
means known to a skilled person, such as, for instance, concentration,
crystallization, precipitation, adsorption, ion exchange,
electrodialysis, bipolar membrane electrodialysis and/or reverse osmosis.
Vitamin C may be further purified as the free acid form or any of its
known salt forms by means of operations such as for instance treatment
with activated carbon, ion exchange, adsorption and elution,
concentration, crystallization, filtration and drying. Specifically, a
first separation of Vitamin C from other components in the harvest stream
might be performed by any suitable combination or repetition of, for
instance, the following methods: two- or three-compartment
electrodialysis, bipolar membrane electrodialysis, reverse osmosis or
adsorption on, for instance, ion exchange resins or non-ionic resins. If
the resulting form of Vitamin C is a salt of L-ascorbic acid, conversion
of the salt form into the free acid form may be performed by for instance
bipolar membrane electrodialysis, ion exchange, simulated moving bed
chromatographic techniques, and the like. Combination of the mentioned
steps, e.g., electrodialysis and bipolar membrane electrodialysis into
one step might be also used as well as combination of the mentioned steps
e.g. several steps of ion exchange by using simulated moving bed
chromatographic methods. Any of these procedures alone or in combination
constitute a convenient means for isolating and purifying the product,
i.e. Vitamin C. The product thus obtained may further be isolated in a
manner such as, e.g. by concentration, crystallization, precipitation,
washing and drying of the crystals and/or further purified by, for
instance, treatment with activated carbon, ion exchange and/or
re-crystallization.

[0066]In a preferred embodiment, Vitamin C is purified from the harvest
stream by a series of downstream processing steps as described above
without having to be transferred to a non-aqueous solution at any time of
this processing, i.e. all steps are performed in an aqueous environment.
Such preferred downstream processing procedure may include for instance
the concentration of the harvest stream coming from the production vessel
by means of two- or three-compartment electrodialysis, conversion of
Vitamin C in its salt form present in the concentrated solution into its
acid form by means of bipolar membrane electrodialysis and/or ion
exchange, purification by methods such as for instance treatment with
activated carbon, ion exchange or non-ionic resins, followed by a further
concentration step and crystallization. These crystals can be separated,
washed and dried. If necessary, the crystals may be again re-solubilized
in water, treated with activated carbon and/or ion exchange resins and
recrystallized. These crystals can then be separated, washed and dried.

[0067]Advantageous embodiments of the invention become evident from the
dependent claims. These and other aspects and embodiments of the present
invention should be apparent to those skilled in the art from the
teachings herein.

[0068]The sequence of the gene comprising a nucleotide sequence according
to SEQ ID NO:1 encoding a SMS 27 protein was determined by sequencing a
genomic clone obtained from Gluconobacter oxydans IFO 3293.

[0069]The invention also relates to a polynucleotide encoding at least a
biologically active fragment or derivative of a SMS 27 polypeptide as
shown in SEQ ID NO:2.

[0070]As used herein, "biologically active fragment or derivative" means a
polypeptide which retains essentially the same biological function or
activity as the polypeptide shown in SEQ ID NO:2. Examples of biological
activity may for instance be enzymatic activity, signaling activity or
antibody reactivity. The term "same biological function" or "functional
equivalent" as used herein means that the protein has essentially the
same biological activity, e.g. enzymatic, signaling antibody reactivity
transcriptionally regulator, as a polypeptide shown in SEQ ID NO:2.

[0071]The polypeptides and polynucleotides of the present invention are
preferably provided in an isolated form, and preferably are purified to
homogeneity.

[0072]The term "isolated" means that the material is removed from its
original environment (e.g., the natural environment if it is naturally
occurring). For example, a naturally-occurring polynucleotide or
polypeptide present in a living microorganism is not isolated, but the
same polynucleotide or polypeptide, separated from some or all of the
coexisting materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such polynucleotides or
polypeptides could be part of a composition and still be isolated in that
such vector or composition is not part of its natural environment.

[0073]An isolated polynucleotide or nucleic acid as used herein may be a
DNA or RNA that is not immediately contiguous with both of the coding
sequences with which it is immediately contiguous (one on the 5'-end and
one on the 3'-end) in the naturally occurring genome of the organism from
which it is derived. Thus, in one embodiment, a nucleic acid includes
some or all of the 5'-non-coding (e.g., promoter) sequences that are
immediately contiguous to the coding sequence. The term "isolated
polynucleotide" therefore includes, for example, a recombinant DNA that
is incorporated into a vector, into an autonomously replicating plasmid
or virus, or into the genomic DNA of a prokaryote or eukaryote, or which
exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment
produced by PCR or restriction endonuclease treatment) independent of
other sequences. It also includes a recombinant DNA that is part of a
hybrid gene encoding an additional polypeptide that is substantially free
of cellular material, viral material, or culture medium (when produced by
recombinant DNA techniques), or chemical precursors or other chemicals
(when chemically synthesized). Moreover, an "isolated nucleic acid
fragment" is a nucleic acid fragment that is not naturally occurring as a
fragment and would not be found in the natural state.

[0074]As used herein, the terms "polynucleotide", "gene" and "recombinant
gene" refer to nucleic acid molecules which may be isolated from
chromosomal DNA, which include an open reading frame encoding a protein,
e.g. G. oxydans IFO 3293 SMS proteins. A polynucleotide may include a
polynucleotide sequence as shown in SEQ ID NO:1 or fragments thereof and
regions upstream and downstream of the gene sequences which may include,
for example, promoter regions, regulator regions and terminator regions
important for the appropriate expression and stabilization of the
polypeptide derived thereof.

[0075]A gene may include coding sequences, non-coding sequences such as
for instance untranslated sequences located at the 3'- and 5'-ends of the
coding region of a gene, and regulatory sequences. Moreover, a gene
refers to an isolated nucleic acid molecule as defined herein. It is
furthermore appreciated by the skilled person that DNA sequence
polymorphisms that lead to changes in the amino acid sequences of SMS
proteins may exist within a population, e.g., the Gluconobacter oxydans
population. Such genetic polymorphism in the SMS 27 gene may exist among
individuals within a population due to natural variation or in cells from
different populations. Such natural variations can typically result in
1-5% variance in the nucleotide sequence of the SMS 27 gene. Any and all
such nucleotide variations and the resulting amino acid polymorphism in
SMS 27 are the result of natural variation and that do not alter the
functional activity of SMS proteins are intended to be within the scope
of the invention.

[0076]As used herein, the terms "polynucleotide" or "nucleic acid
molecule" are intended to include DNA molecules (e.g., cDNA or genomic
DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA
generated using nucleotide analogs. The nucleic acid molecule may be
single-stranded or double-stranded, but preferably is double-stranded
DNA. The nucleic acid may be synthesized using oligonucleotide analogs or
derivatives (e.g., inosine or phosphorothioate nucleotides). Such
oligonucleotides may be used, for example, to prepare nucleic acids that
have altered base-pairing abilities or increased resistance to nucleases.

[0077]The sequence information as provided herein should not be so
narrowly construed as to require inclusion of erroneously identified
bases. The specific sequences disclosed herein may be readily used to
isolate the complete gene from a recombinant or non-recombinant
microorganism capable of converting a given carbon source directly into
Vitamin C and/or 2-KGA, in particular Gluconobacter oxydans, preferably
Gluconobacter oxydans IFO 3293 which in turn may easily be subjected to
further sequence analyses thereby identifying sequencing errors.

[0078]Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated DNA
sequencer and all amino acid sequences of polypeptides encoded by DNA
molecules determined herein were predicted by translation of a DNA
sequence determined as above. Therefore, as is known in the art for any
DNA sequence determined by this automated approach, any nucleotide
sequence determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to the
actual nucleotide sequence of the sequenced DNA molecule. The actual
sequence may be more precisely determined by other approaches including
manual DNA sequencing methods well known in the art. As is also known in
the art, a single insertion or deletion in a determined nucleotide
sequence compared to the actual sequence will cause a frame shift in
translation of the nucleotide sequence such that the predicted amino acid
sequence encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the sequenced
DNA molecule, beginning at the point of such an insertion or deletion.

[0079]The person skilled in the art is capable of identifying such
erroneously identified bases and knows how to correct for such errors.

[0080]A nucleic acid molecule according to the invention may comprise only
a portion or a fragment of the nucleic acid sequence provided by the
present invention, such as for instance the sequence shown in SEQ ID
NO:1, for example a fragment which may be used as a probe or primer such
as for instance SEQ ID NO:3 or SEQ ID NO:4 or a fragment encoding a
portion of a protein according to the invention. The nucleotide sequence
determined from the cloning of the SMS 27 gene allows for the generation
of probes and primers designed for use in identifying and/or cloning
other SMS 27 family members, as well as SMS 27 homologues from other
species. The probe/primer typically comprises substantially purified
oligonucleotides which typically comprises a region of nucleotide
sequence that hybridizes preferably under highly stringent conditions to
at least about 12 or 15, preferably about 18 or 20, more preferably about
22 or 25, even more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or
75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ
ID NO:1 or a fragment or derivative thereof.

[0081]A nucleic acid molecule encompassing all or a portion of the nucleic
acid sequence of SEQ ID NO:1 may be also isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed based
upon the sequence information contained herein.

[0082]A nucleic acid of the invention may be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers according to standard PCR amplification techniques. The nucleic
acid thus amplified may be cloned into an appropriate vector and
characterized by DNA sequence analysis.

[0083]Fragments of a polynucleotide according to the invention may also
comprise polynucleotides not encoding functional polypeptides. Such
polynucleotides may function as probes or primers for a PCR reaction.

[0084]Nucleic acids according to the invention irrespective of whether
they encode functional or non-functional polypeptides, may be used as
hybridization probes or polymerase chain reaction (PCR) primers. Uses of
the nucleic acid molecules of the present invention that do not encode a
polypeptide having a SMS 27 activity include, inter alia, (1) isolating
the gene encoding the protein of the present invention, or allelic
variants thereof from a cDNA library, e.g., from other organisms than
Gluconobacter oxydans and (2) Northern blot analysis for detecting
expression of mRNA of said protein in specific cells or (3) use in
enhancing and/or improving the function or activity of homologous SMS 27
genes in said other organisms.

[0085]Probes based on the nucleotide sequences provided herein may be used
to detect transcripts or genomic sequences encoding the same or
homologous proteins for instance in other organisms. Nucleic acid
molecules corresponding to natural variants and non-G. oxydans homologues
of the G. oxydans SMS 27 DNA of the invention which are also embraced by
the present invention may be isolated based on their homology to the G.
oxydans SMS 27 nucleic acid disclosed herein using the G. oxydans DNA, or
a portion thereof, as a hybridization probe according to standard
hybridization techniques, preferably under highly stringent hybridization
conditions.

[0086]In preferred embodiments, the probe further comprises a label group
attached thereto, e.g., the label group can be a radioisotope, a
fluorescent compound, an enzyme, or an enzyme cofactor.

[0087]Homologous gene sequences may be isolated, for example, by
performing PCR using two degenerate oligonucleotide primer pools designed
on the basis of nucleotide sequences as taught herein.

[0088]The template for the reaction may be cDNA obtained by reverse
transcription of mRNA prepared from strains known or suspected to express
a polynucleotide according to the invention. The PCR product may be
subcloned and sequenced to ensure that the amplified sequences represent
the sequences of a new nucleic acid sequence as described herein, or a
functional equivalent thereof.

[0089]The PCR fragment may then be used to isolate a full length cDNA
clone by a variety of known methods. For example, the amplified fragment
may be labeled and used to screen a bacteriophage or cosmid cDNA library.
Alternatively, the labeled fragment may be used to screen a genomic
library.

[0090]PCR technology can also be used to isolate full-length cDNA
sequences from other organisms. For example, RNA may be isolated,
following standard procedures, from an appropriate cellular or tissue
source. A reverse transcription reaction may be performed on the RNA
using an oligonucleotide primer specific for the most 5'-end of the
amplified fragment for the priming of first strand synthesis.

[0091]The resulting RNA/DNA hybrid may then be "tailed" (e.g., with
guanines) using a standard terminal transferase reaction, the hybrid may
be digested with RNaseH, and second strand synthesis may then be primed
(e.g., with a poly-C primer). Thus, cDNA sequences upstream of the
amplified fragment may easily be isolated. For a review of useful cloning
strategies, see e.g., Sambrook et al., supra; and Ausubel et al., supra.

[0092]Also, nucleic acids encoding other SMS 27 family members, which thus
have a nucleotide sequence that differs from a nucleotide sequence
according to SEQ ID NO:1, are within the scope of the invention.
Moreover, nucleic acids encoding SMS 27 proteins from different species
which thus may have a nucleotide sequence which differs from a nucleotide
sequence shown in SEQ ID NO:1 are within the scope of the invention.

[0093]The invention also relates to an isolated polynucleotide
hybridisable under stringent conditions, preferably under highly
stringent conditions, to a polynucleotide as of the present invention,
such as for instance a polynucleotide shown in SEQ ID NO:1.
Advantageously, such polynucleotide may be obtained from a microorganism
capable of converting a given carbon source directly into Vitamin C, in
particular Gluconobacter oxydans, preferably Gluconobacter oxydans IFO
3293.

[0094]As used herein, the term "hybridizing" is intended to describe
conditions for hybridization and washing under which nucleotide sequences
at least about 50%, at least about 60%, at least about 70%, more
preferably at least about 80%, even more preferably at least about 85% to
90%, most preferably at least 95% homologous to each other typically
remain hybridized to each other.

[0096]A preferred, non-limiting example of stringent hybridization
conditions are hybridization in 6× sodium chloride/sodium citrate
(SSC) at about 45° C., followed by one or more washes in
1×SSC, 0.1% SDS at 50° C., preferably at 55° C., more
preferably at 60° C. and even more preferably at 65° C.

[0097]Highly stringent conditions include incubations at 42° C. for
a period of several days, such as 2-4 days, using a labeled DNA probe,
such as a digoxigenin (DIG)-labeled DNA probe, followed by one or more
washes in 2×SSC, 0.1% SDS at room temperature and one or more
washes in 0.5×SSC, 0.1% SDS or 0.1×SSC, 0.1% SDS at
65-68° C. In particular, highly stringent conditions include, for
example, 2 h to 4 days incubation at 42° C. using a DIG-labeled
DNA probe (prepared by e.g. using a DIG labeling system; Roche
Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as
DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100 μg/ml
salmon sperm DNA, or a solution comprising 50% formamide, 5×SSC
(150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate,
0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics
GmbH), followed by washing the filters twice for 5 to 15 minutes in
2×SSC and 0.1% SDS at room temperature and then washing twice for
15-30 minutes in 0.5×SSC and 0.1% SDS or 0.1×SSC and 0.1% SDS
at 65-68° C.

[0098]Preferably, an isolated nucleic acid molecule of the invention that
hybridizes under preferably highly stringent conditions to a nucleotide
sequence of the invention corresponds to a naturally-occurring nucleic
acid molecule. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide sequence
that occurs in nature (e.g., encodes a natural protein). In one
embodiment, the nucleic acid encodes a natural G. oxydans SMS 27 protein.

[0099]The skilled artisan will know which conditions to apply for
stringent and highly stringent hybridization conditions. Additional
guidance regarding such conditions is readily available in the art, for
example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995,
Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.). Of
course, a polynucleotide which hybridizes only to a poly (A) sequence
(such as the 3'-terminal poly (A) tract of mRNAs), or to a complementary
stretch of T (or U) residues, would not be included in a polynucleotide
of the invention used to specifically hybridize to a portion of a nucleic
acid of the invention, since such a polynucleotide would hybridize to any
nucleic acid molecule containing a poly (A) stretch or the complement
thereof (e.g., practically any double-stranded cDNA clone).

[0100]In a typical approach, genomic DNA or cDNA libraries constructed
from other organisms, e.g. microorganisms capable of converting a given
carbon source directly into Vitamin C, in particular other Gluconobacter
species may be screened.

[0101]For example, Gluconobacter strains may be screened for homologous
polynucleotides by Southern and/or Northern blot analysis. Upon detection
of transcripts homologous to polynucleotides according to the invention,
DNA libraries may be constructed from RNA isolated from the appropriate
strain, utilizing standard techniques well known to those of skill in the
art. Alternatively, a total genomic DNA library may be screened using a
probe hybridisable to a polynucleotide according to the invention.

[0102]A nucleic acid molecule of the present invention, such as for
instance a nucleic acid molecule shown in SEQ ID NO:1 or a fragment or
derivative thereof, may be isolated using standard molecular biology
techniques and the sequence information provided herein. For example,
using all or portion of the nucleic acid sequence shown in SEQ ID NO:1 as
a hybridization probe, nucleic acid molecules according to the invention
may be isolated using standard hybridization and cloning techniques
(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.
Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989).

[0103]Furthermore, oligonucleotides corresponding to or hybridisable to
nucleotide sequences according to the invention may be prepared by
standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0104]The terms "homology" or "percent identity" are used interchangeably
herein. For the purpose of this invention, it is defined here that in
order to determine the percent identity of two amino acid sequences or of
two nucleic acid sequences, the sequences are aligned for optimal
comparison purposes (e.g., gaps may be introduced in the sequence of a
first amino acid or nucleic acid sequence for optimal alignment with a
second amino or nucleic acid sequence). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide positions
are then compared. When a position in the first sequence is occupied by
the same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a function of
the number of identical positions shared by the sequences (i.e., %
identity=number of identical positions/total number of positions (i.e.,
overlapping positions)×100). Preferably, the two sequences are the
same length.

[0105]The skilled person will be aware of the fact that several different
computer programs are available to determine the homology between two
sequences. For instance, a comparison of sequences and determination of
percent identity between two sequences may be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent identity
between two amino acid sequences is determined using the Needleman and
Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package (available
at http://www.accelrys.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight
of 1, 2, 3, 4, 5 or 6. The skilled person will appreciate that all these
different parameters will yield slightly different results but that the
overall percentage identity of two sequences is not significantly altered
when using different algorithms.

[0106]In yet another embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.accelrys.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a
length weight of 1, 2, 3, 4, 5 or 6. In another embodiment, the percent
identity between two amino acid or nucleotide sequences is determined
using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)
which has been incorporated into the ALIGN program (version 2.0)
(available at http://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0107]The nucleic acid and protein sequences of the present invention may
further be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches may be performed using the BLASTN and BLASTX
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.
215:403-10. BLAST nucleotide searches may be performed with the BLASTN
program, score=100, word length=12 to obtain nucleotide sequences
homologous to the nucleic acid molecules of the present invention. BLAST
protein searches may be performed with the BLASTX program, score=50, word
length=3 to obtain amino acid sequences homologous to the protein
molecules of the present invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST may be utilized as described in
Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters of the
respective programs (e.g., BLASTX and BLASTN) may be used. See
http://www.ncbi.nlm.nih.gov.

[0108]In another preferred embodiment, an isolated nucleic acid molecule
of the invention comprises a nucleic acid molecule which is the
complement of a nucleotide sequence as of the present invention, such as
for instance the sequence shown in SEQ ID NO:1. A nucleic acid molecule,
which is complementary to a nucleotide sequence disclosed herein, is one
that is sufficiently complementary to a nucleotide sequence shown in SEQ
ID NO:1 such that it may hybridize to said nucleotide sequence thereby
forming a stable duplex.

[0109]In a further preferred embodiment, a nucleic acid of the invention
as shown in SEQ ID NO:1 or the complement thereof contains at least one
mutation leading to a gene product with modified function/activity. The
at least one mutation may be introduced by methods described herein. In
one aspect, the at least one mutation leads to a SMS 27 protein whose
function compared to the wild type counterpart is completely or partially
destroyed. Methods for introducing such mutations are well known in the
art.

[0110]The term "reduction" of activity as used herein encompasses
decreasing activity of one or more polypeptides in the producing
organism, which in turn are encoded by the corresponding polynucleotides
described herein. There are a number of methods available in the art to
accomplish reduction of activity of a given protein, in this case the SMS
27 protein. In general, the specific activity of a protein may be
decreased or the copy number of the protein may be decreased.

[0111]To facilitate such a decrease, the copy number of the genes
corresponding to the polynucleotides described herein may be decreased,
such as for instance by underexpression or disruption of a gene. A gene
is said to be "underexpressed" if the level of transcription of said gene
is reduced in comparison to the wild type gene. This may be measured by
for instance Northern blot analysis quantifying the amount of mRNA as an
indication for gene expression. As used herein, a gene is underexpressed
if the amount of generated mRNA is decreased by at least 1%, 2%, 5% 10%,
25%, 50%, 75%, 100%, 200% or even more than 500%, compared to the amount
of mRNA generated from a wild-type gene. Alternatively, a weak promoter
may be used to direct the expression of the polynucleotide. In another
embodiment, the promoter, regulatory region and/or the ribosome binding
site upstream of the gene can be altered to achieve the down-expression.
The expression may also be reduced by decreasing the relative half-life
of the messenger RNA. In another embodiment, the activity of the
polypeptide itself may be decreased by employing one or more mutations in
the polypeptide amino acid sequence, which decrease the activity. For
example, altering the affinity of the polypeptide for its corresponding
substrate may result in reduced activity. Likewise, the relative
half-life of the polypeptide may be decreased. In either scenario, that
being reduced gene expression or reduced activity, the reduction may be
achieved by altering the composition of the cell culture media and/or
methods used for culturing. "Reduced expression" or "reduced activity" as
used herein means a decrease of at least 5%, 10%, 25%, 50%, 75%, 100%,
200% or even more than 500%, compared to a wild-type protein,
polynucleotide, gene; or the activity and/or the concentration of the
protein present before the polynucleotides or polypeptides are reduced.
The activity of the SMS 27 protein may also be reduced by contacting the
protein with a specific or general inhibitor of its activity. The terms
"reduced activity", "decreased or abolished activity" are used
interchangeably herein.

[0112]Another aspect of the invention pertains to vectors, containing a
nucleic acid encoding a protein according to the invention or a
functional equivalent or portion thereof. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a viral
vector, wherein additional DNA segments may be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having a
bacterial origin of replication). Other vectors are integrated into the
genome of a host cell upon introduction into the host cell, and thereby
are replicated along with the host genome.

[0113]The recombinant vectors of the invention comprise a nucleic acid of
the invention in a form suitable for expression of the nucleic acid in a
host cell, which means that the recombinant expression vector includes
one or more regulatory sequences, selected on the basis of the host cells
to be used for expression, which is operatively linked to the nucleic
acid sequence to be expressed. Within a recombinant expression vector,
"operatively linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which allows
for expression of the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a host cell when the vector is
introduced into the host cell). The term "regulatory sequence" is
intended to include promoters, enhancers and other expression control
elements (e.g., attenuator). Such regulatory sequences are described, for
example, in Goeddel; Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990). Regulatory sequences
include those which direct constitutive or inducible expression of a
nucleotide sequence in many types of host cells and those which direct
expression of the nucleotide sequence only in a certain host cell (e.g.
tissue-specific regulatory sequences). It will be appreciated by those
skilled in the art that the design of the expression vector can depend on
such factors as the choice of the host cell to be transformed, the level
of expression of protein desired, etc. The expression vectors of the
invention may be introduced into host cells to thereby produce proteins
or peptides, encoded by nucleic acids as described herein, including, but
not limited to, mutant proteins, fragments thereof, variants or
functional equivalents thereof, and fusion proteins, encoded by a nucleic
acid as described herein, e.g., SMS 27 proteins, mutant forms of SMS 27
proteins, fusion proteins and the like.

[0114]The recombinant expression vectors of the invention may be designed
for expression of SMS 27 proteins in a suitable microorganism. For
example, a protein according to the invention may be expressed in
bacterial cells such as strains belonging to the genera Gluconobacter,
Gluconacetobacter or Acetobacter. Expression vectors useful in the
present invention include chromosomal-, episomal- and virus-derived
vectors e.g., vectors derived from bacterial plasmids, bacteriophage, and
vectors derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids.

[0115]The DNA insert may be operatively linked to an appropriate promoter,
which may be either a constitutive or inducible promoter. The skilled
person will know how to select suitable promoters. The expression
constructs may contain sites for transcription initiation, termination,
and, in the transcribed region, a ribosome binding site for translation.
The coding portion of the mature transcripts expressed by the constructs
may preferably include an initiation codon at the beginning and a
termination codon appropriately positioned at the end of the polypeptide
to be translated.

[0116]Vector DNA may be introduced into suitable host cells via
conventional transformation or transfection techniques. As used herein,
the terms "transformation", "transconjugation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, transduction, infection, lipofection,
cationic lipidmediated transfection or electroporation. Suitable methods
for transforming or transfecting host cells may be found in Sambrook, et
al. (supra), Davis et al., Basic Methods in Molecular Biology (1986) and
other laboratory manuals.

[0117]In order to identify and select cells which have integrated the
foreign DNA into their genome, a gene that encodes a selectable marker
(e.g., resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those that confer resistance to drugs, such as kanamycin,
tetracycline, ampicillin and streptomycin. A nucleic acid encoding a
selectable marker is preferably introduced into a host cell on the same
vector as that encoding a protein according to the invention or can be
introduced on a separate vector such as, for example, a suicide vector,
which cannot replicate in the host cells. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will survive,
while the other cells die).

[0118]The invention provides also an isolated polypeptide having the amino
acid sequence shown in SEQ ID NO:2 or an amino acid sequence obtainable
by expressing a polynucleotide of the present invention, such as for
instance a polynucleotide sequence shown in SEQ ID NO:1 in an appropriate
host.

[0119]Polypeptides according to the invention may contain only
conservative substitutions of one or more amino acids in the amino acid
sequence represented by SEQ ID NO:2 or substitutions, insertions or
deletions of non-essential amino acids. Accordingly, a non-essential
amino acid is a residue that may be altered in the amino acid sequences
shown in SEQ ID NO:2 without substantially altering the biological
function. For example, amino acid residues that are conserved among the
proteins of the present invention, are predicted to be particularly
unamenable to alteration. Furthermore, amino acids conserved among the
proteins according to the present invention and other SMS 27 proteins are
not likely to be amenable to alteration.

[0120]The term "conservative substitution" is intended to mean that a
substitution in which the amino acid residue is replaced with an amino
acid residue having a similar side chain. These families are known in the
art and include amino acids with basic side chains (e.g., lysine,
arginine and histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains
(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine).

[0121]As mentioned above, the polynucleotides of the invention may be
utilized in the genetic engineering of a suitable host cell to make it
better and more efficient in the fermentation, for example in a direct
fermentation process for Vitamin C.

[0122]According to the invention a genetically engineered/recombinantly
produced host cell (also referred to as recombinant cell or transformed
cell) carrying such a modified polynucleotide wherein the function of the
linked protein is significantly modified in comparison to a wild-type
cell such that the yield, production and/or efficiency of production of
one or more fermentation products such as Vitamin C is improved. The host
cell may be selected from a microorganism capable of directly producing
one or more fermentation products such as for instance Vitamin C from a
given carbon source, in particular Gluconobacter oxydans, preferably G.
oxydans IFO 3293.

[0123]A "transformed cell" or "recombinant cell" is a cell into which (or
into an ancestor of which) has been introduced, by means of recombinant
DNA techniques, a nucleic acid according to the invention, or wherein the
activity of the SMS 27 protein has been decreased or abolished. Suitable
host cells include cells of microorganisms capable of producing a given
fermentation product, e.g., converting a given carbon source directly
into Vitamin C. In particular, these include strains from the genera
Pseudomonas, Pantoea, Escherichia, Corynebacterium, Ketogulonicigenium
and acetic acid bacteria like e.g., Gluconobacter, Acetobacter or
Gluconacetobacter, preferably Acetobacter sp., Acetobacter aceti,
Gluconobacter frateurii, Gluconobacter cerinus, Gluconobacter
thailandicus, Gluconobacter oxydans, more preferably G. oxydans, most
preferably G. oxydans IFO 3293.

[0125]Inhibition or prevention of gene expression may also be achieved by
modifying the SMS 27 gene, e.g., by introducing one or more mutations
into the SMS 27gene wherein said modification leads to a SMS 27 protein
with a function which is significantly decreased in comparison to the
wild-type protein.

[0126]Therefore, in one other embodiment, the polynucleotide carrying the
at least one mutation is derived from a polynucleotide as represented by
SEQ ID NO:1 or equivalents thereof

[0127]A mutation as used herein may be any mutation leading to a less
functional or unstable polypeptide, e.g. less functional or unstable SMS
27 gene products. This may include for instance an alteration in the
genome of a microorganism, which interferes with the synthesis of SMS 27
or leads to the expression of a SMS 27 protein with an altered amino acid
sequence whose function compared with the wild type counterpart having a
non-altered amino acid sequence is completely or partially destroyed. The
interference may occur at the transcriptional, translational or
post-translational level.

[0128]The alteration in the genome of the microorganism may be obtained
e.g. by replacing through a single or double crossover recombination a
wild type DNA sequence by a DNA sequence containing the alteration. For
convenient selection of transformants of the microorganism with the
alteration in its genome the alteration may, e.g. be a DNA sequence
encoding an antibiotic resistance marker or a gene complementing a
possible auxotrophy of the microorganism. Mutations include, but are not
limited to, deletion-insertion mutations. An example of such an
alteration includes a gene disruption, i.e. a perturbation of a gene such
that the product that is normally produced from this gene is not produced
in a functional form. This could be due to a complete deletion, a
deletion and insertion of a selective marker, an insertion of a selective
marker, a frameshift mutation, an in-frame deletion, or a point mutation
that leads to premature termination. In some of these cases the entire
mRNA for the gene is absent, in others the amount of mRNA produced
varies. In all cases the polypeptide encoded by said gene is not produced
in a functional form, either absent or in a mutated form, such as e.g. a
protein having reduced activity as defined herein.

[0129]An alteration in the genome of the microorganism leading to a less
or non-functional polypeptide may also be obtained by randomly
mutagenizing the genome of the microorganism using e.g. chemical
mutagens, radiation or transposons and selecting or screening for mutants
which are better or more efficient producers of one or more fermentation
products. Standard methods for screening and selection are known to the
skilled person.

[0130]In a specific embodiment, it is desired to knockout the SMS 27 gene
of the present invention, i.e., wherein its gene expression is
artificially suppressed in order to improve the yield, productivity,
and/or efficiency of production of the fermentation product when
introduced into a suitable host cell. Methods of providing knockouts as
well as microorganisms carrying such suppressed genes are well known in
the art. The suppression of the endogenous SMS 27 gene may be induced by
deleting at least a part of the gene or the regulatory region thereof. As
used herein, "suppression of the gene expression" includes complete and
partial suppression, as well as suppression under specific conditions and
also suppression of the expression of either one of the two alleles.

[0131]In order to create a knockout microorganism in which the expression
of the SMS 27 gene is artificially suppressed, first the SMS 27 gene may
be cloned and then a vector for homologous recombination may be
constructed by using the gene to inactivate the endogenous SMS 27 gene in
the target microorganism. The vector for homologous recombination then
contains a nucleic acid sequence designed to inactivate the endogenous
SMS 27 gene in the target microorganism. Such a nucleic acid may be for
instance a nucleic acid sequence of the SMS 27 gene or the regulatory
region thereof, such as the existing flanking region of the gene to be
inactivated (in cis), or existing separately (in trans), containing at
least a partial deletion, or alternatively it may be a nucleic acid
sequence of the SMS 27 gene or the regulatory region thereof containing
other genes. A gene which can also function as a marker is preferably
selected as the gene to be inserted into the SMS 27 gene or the
regulatory region thereof. The insert genes to be used include for
instance drug-resistance genes as defined above. There is no particular
limitation on the position where the genes may be inserted in the SMS 27
gene, as long as the insertion at that position results in the
suppression of the expression of the endogenous SMS 27 gene in the
target. To avoid polar effects of the insertion, in-frame silent
deletions can be introduced by using, for example, the sacB system or
long-flanking homology PCR. These techniques are well known to the person
skilled in the art.

[0132]The aforementioned mutagenesis strategies for SMS 27 proteins may
result in increased yields of a desired compound in particular Vitamin C
and/or 2-KGA. This list is not meant to be limiting; variations on these
mutagenesis strategies will be readily apparent to one of ordinary skill
in the art. By these mechanisms, the nucleic acid and protein molecules
of the invention may be utilized to generate microorganisms such as
Gluconobacter oxydans or related strains of bacteria expressing mutated
SMS 27 nucleic acid and protein molecules such that the yield,
productivity, and/or efficiency of production of a desired compound such
as Vitamin C and/or 2-KGA is improved.

[0133]In connection with the above process using a microorganism, in one
aspect, the process of the present invention leads to yields of Vitamin C
which are at least about more than 5.7 g/l, such as 10 g/l, 20 g/l, 50
g/l, 100 g/l, 200 g/l, 300 g/l, 400 g/l or more than 600 g/l. In one
embodiment, the yield of Vitamin C produced by the process of the present
invention is in the range of from about more than 5.7 to about 600 g/l.
The yield of Vitamin C refers to the concentration of Vitamin C in the
harvest stream coming directly out of the production vessel, i.e. the
cell-free supernatant comprising the Vitamin C.

[0134]In one aspect of the invention, microorganisms (in particular from
the genera of Gluconobacter, Gluconacetobacter and Acetobacter) are
provided that are able to directly produce Vitamin C from a suitable
carbon source like D-sorbitol and/or L-sorbose. When measured for
instance in a resting cell method after an incubation period of 20 hours,
these organisms were found to be able to produce Vitamin C directly from
D-sorbitol or L-sorbose, even up to a level of 280 mg/l and 670 mg/l
respectively. In another aspect of the invention, a microorganism is
provided capable of directly producing Vitamin C in quantities of 300
mg/l when starting from D-sorbitol or more or 800 mg/l or more when
starting from L-sorbose, respectively when for instance measured in a
resting cell method after an incubation period of 20 hours. Such may be
achieved by decreasing or abolishing the activity of a SMS polypeptide,
preferably a SMS 27 polypeptide. The yield of Vitamin C produced from
D-sorbitol may even be as high as 400, 600, 1000 mg/l or even exceed 1.5,
2, 4, 10, 20, 50 g/l. The yield of Vitamin C produced from L-sorbose may
even be as high as 1000 mg/l or even exceed 1.5, 2, 4, 10, 20, 50 μl.
Preferably, these amounts of Vitamin C can be achieved when measured by
resting cell method after an incubation period of 20 hours.

[0135]As used herein, measurement in a "resting cell method" comprises (i)
growing the cells by means of any method well know to the person skilled
in the art, (ii) harvesting the cells from the growth broth, and (iii)
incubating the harvested cells in a medium containing the substrate which
is to be converted into the desired product, e.g. Vitamin C, under
conditions where the cells do not grow any longer, i.e. there is no
increase in the amount of biomass during this so-called conversion step.
A more general description of the resting cell method is described for
instance in WO 2005/017159 and in the following paragraphs.

[0136]In one aspect of the invention, microorganisms (in particular from
the genera of Gluconobacter, Gluconacetobacter and Acetobacter) are
provided that are able to directly produce 2-KGA from a suitable carbon
source like D-sorbitol and/or L-sorbose. When measured for instance by
the method as of Example 4, these organisms were found to be able to
produce 2-KGA directly from D-sorbitol or L-sorbose in amounts of about
0.5 to 0.7 g/l. In another aspect of the invention, a microorganism is
provided capable of directly producing 2-KGA in quantities of about 7, 8,
9, 10 g/l or more or even about 50, 60, 70, 80, 90, 100 g/l or more when
starting from L-sorbose. Such may be achieved by decreasing or even
abolishing the activity of a SMS polypeptide, preferably a SMS 27
polypeptide in G. oxydans IFO 3293.

[0137]The recombinant microorganism carrying e.g. a modified SMS 27 gene
and which is able to produce the fermentation product in significantly
higher yield, productivity, and/or efficiency may be cultured in an
aqueous medium supplemented with appropriate nutrients under aerobic
conditions as described above.

[0138]The nucleic acid molecules, polypeptides, vectors, primers, and
recombinant microorganisms described herein may be used in one or more of
the following methods: identification of Gluconobacter oxydans and
related organisms; mapping of genomes of organisms related to
Gluconobacter oxydans; identification and localization of Gluconobacter
oxydans sequences of interest; evolutionary studies; determination of SMS
27 protein regions required for function; modulation of a SMS 27 protein
activity or function; modulation of the activity of a SMS pathway; and
modulation of cellular production of a desired compound, such as Vitamin
C and/or 2-KGA.

[0139]The invention provides methods for screening molecules which
modulate the activity of a SMS 27 protein, either by interacting with the
protein itself or a substrate or binding partner of the SMS 27 protein,
or by modulating the transcription or translation of a SMS 27 nucleic
acid molecule of the invention. In such methods, a microorganism
expressing one or more SMS 27 proteins of the invention is contacted with
one or more test compounds, and the effect of each test compound on the
activity or level of expression of the SMS 27 protein is assessed.

[0140]In general, the biological, enzymatic or other activity of SMS
proteins can be measured by methods well known to a skilled person, such
as, for example, by incubating a cell fraction containing the SMS protein
in the presence of its substrate, electron acceptor(s) or donor(s)
including phenazine methosulfate (PMS), dichlorophenol-indophenol (DCIP),
NAD, NADH, NADP, NADPH, which consumption can be directly or indirectly
measured by photometric, colorimetric or fluorimetric methods, and other
inorganic components which might be relevant for the development of the
activity. Thus, for example, the activity of membrane-bound D-sorbitol
dehydrogenase can be measured in an assay where membrane fractions
containing this enzyme are incubated in the presence of phosphate buffer
at pH 6, D-sorbitol and the artificial electron acceptors DCIP and PMS.
The rate of consumption of DCIP can be measured at 600 nm, and is
directly proportional to the D-sorbitol dehydrogenase activity present in
the membrane fraction.

[0141]The biological, enzymatic or other activity of SMS proteins, in
particular the SMS 27 protein, can be measured by methods well known to a
skilled person, such as, for example, by determining the expression of
genes known to be under the control of the SMS 27 protein by methods
known to those skilled in the art, such as for instance Northern Blot,
transcriptional fusion analysis, microarray analysis, etc. Binding of SMS
27 protein to target promoter regions can be demonstrated by DNA
footprint experiments, gel-shift assays and the like.

[0142]It may be evident from the above description that the fermentation
product of the methods according to the invention may not be limited to
Vitamin C alone. The "desired compound" or "fermentation product" as used
herein may be any natural product of Gluconobacter oxydans, which
includes the final products and intermediates of biosynthesis pathways,
such as for example L-sorbose, L-sorbosone, D-gluconate,
2-keto-D-gluconate, 5-keto-D-gluconate, 2,5-diketo-D-gluconate and
2-keto-L-gluconate (2-KGA), in particular the biosynthetic generation of
Vitamin C.

[0143]Thus, the present invention is directed to the use of a
polynucleotide, polypeptide, vector, primer and recombinant microorganism
as described herein in the production of Vitamin C and/or 2-KGA, i.e.,
the direct conversion of a carbon source into Vitamin C and/or 2-KGA. In
a preferred embodiment, a modified polynucleotide, polypeptide, vector
and recombinant microorganism as described herein is used for improving
the yield, productivity, and/or efficiency of the production of Vitamin C
and/or 2-KGA.

[0144]The terms "production" or "productivity" are art-recognized and
include the concentration of the fermentation product (for example,
Vitamin C and/or 2-KGA) formed within a given time and a given
fermentation volume (e.g., kg product per hour per liter). The term
"efficiency of production" includes the time required for a particular
level of production to be achieved (for example, how long it takes for
the cell to attain a particular rate of output of a fermentation
product). The term "yield" is art-recognized and includes the efficiency
of the conversion of the carbon source into the product (i.e., Vitamin
C). This is generally written as, for example, kg product per kg carbon
source. By "increasing the yield and/or production/productivity" of the
compound it is meant that the quantity of recovered molecules, or of
useful recovered molecules of that compound in a given amount of culture
over a given amount of time is increased. The terms "biosynthesis" or a
"biosynthetic pathway" are art-recognized and include the synthesis of a
compound, preferably an organic compound, by a cell from intermediate
compounds in what may be a multistep and highly regulated process. The
language "metabolism" is art-recognized and includes the totality of the
biochemical reactions that take place in an organism. The metabolism of a
particular compound, then, (e.g., the metabolism of an amino acid such as
glycine) comprises the overall biosynthetic, modification, and
degradation pathways in the cell related to this compound. The language
"transport" or "import" is art-recognized and includes the facilitated
movement of one or more molecules across a cellular membrane through
which the molecule would otherwise either be unable to pass or be passed
inefficiently.

[0145]Vitamin C as used herein may be any chemical form of L-ascorbic acid
found in aqueous solutions, such as for instance undissociated, in its
free acid form or dissociated as an anion. The solubilized salt form of
L-ascorbic acid may be characterized as the anion in the presence of any
kind of cations usually found in fermentation supernatants, such as for
instance potassium, sodium, ammonium, or calcium. Also included may be
isolated crystals of the free acid form of L-ascorbic acid. On the other
hand, isolated crystals of a salt form of L-ascorbic acid are called by
their corresponding salt name, i.e. sodium ascorbate, potassium
ascorbate, calcium ascorbate and the like.

[0146]In one preferred embodiment, the present invention is related to a
process for the production of Vitamin C wherein a nucleotide according to
the invention as described above is inactivated in a suitable
microorganism by methods described above, the recombinant microorganism
is cultured under conditions that allow the production of Vitamin C in
high productivity, yield, and/or efficiency, the produced fermentation
product is isolated from the culture medium and optionally further
purified.

[0147]This invention is further illustrated by the following examples
which should not be construed as limiting. The contents of all
references, patent applications, patents and published patent
applications, cited throughout this application are hereby incorporated
by reference.

EXAMPLES

Example 1

Preparation of Chromosomal DNA and Amplification of DNA Fragment by PCR

[0149]A DNA fragment was prepared by PCR with the chromosomal DNA prepared
above and a set of primers, Pf (SEQ ID NO:3) and Pr (SEQ ID NO:4). For
the reaction, the Expand High Fidelity PCR kit (Roche Diagnostics) and 10
ng of the chromosomal DNA was used in total volume of 100 μL according
to the supplier's instruction to have the PCR product containing SMS 27
DNA sequence (SEQ ID NO:1). The PCR product was recovered from the
reaction and its correct sequence confirmed.

Example 2

Disruption of the SMS 27 Gene in G. oxydans IFO 3293

[0150]In order to construct a knockout mutant of the SMS 27 gene,
Long-Flanking Homology (LFH) PCR was used to construct a clean
deletion-insertion mutation. Firstly, the upstream and downstream regions
flanking the SMS 27 gene were amplified by PCR using the respective
primer pairs SMS 27LFH+1 (SEQ ID NO:5)/SMS 27 KmLFH-1 (SEQ ID NO:6) and
SMS 27 KmLFH+1 (SEQ ID NO:7)/SMS 27LFH-1 (SEQ ID NO:8). G. oxydans IFO
3293 genomic DNA was used as a template and the reaction conditions
consisted of 30 cycles of denaturation at 95° C. for 1 min,
annealing at 50° C. for 1 min and extension at 72° C. for
1.5 min. In both cases, the Herculase DNA polymerase (Stratagene) was
used to minimize PCR-generated errors. The kanamycin-resistance cassette
was amplified using pUC4K plasmid DNA (Amersham Bioscience, accession No.
X06404) as a template and primer pair SMS 27 Km-1 (SEQ ID NO:9)/SMS 27
Km+1 (SEQ ID NO:10) to generate a 1.3-kb fragment. The reaction
conditions were as above. The three products were gel-purified, mixed and
used in the second round PCR reaction with the flanking primers SMS
27LFH+1/SMS 27LFH-1 to generate a product of 2.6-kb. The reaction
conditions for the second round reaction consisted of 94° C., 2
min, then 10 cycles of [94° C., 30 sec, 63° C., 30 sec,
68° C., 6 min], followed by 20 cycles of [94° C., 30 sec,
63° C., 30 sec, 68° C., 6 min with an additional 20 sec per
cycle] and a final extension at 68° C. for 10 min.

[0152]Cells of G. oxydans IFO 3293 and G. oxydans IFO 3293-SMS 27::Km were
plated firstly on MB medium for three days. Then cells were scraped off
these plates and spread onto No. 3BD medium containing 7% sorbitol and
grown for 3 days at 30° C. These cells were used in resting-cell
reactions with 2% sorbitol as substrate and a cell density of
OD600=10.

[0158]Genomic DNA preparations are digested with restriction enzymes such
as EcoRI or HindIII, and 1 μg of the DNA fragments are separated by
agarose gel electrophoresis (1% agarose). The gel is treated with 0.25 N
HCl for 15 min and then 0.5 N NaOH for 30 min, and then blotted onto
nitrocellulose or a nylon membrane with Vacuum Blotter Model 785 (BIO-RAD
Laboratories AG, Switzerland) according to the instruction of the
supplier. The resulting blot is then brought into contact/hybridized with
a solution wherein the probe, such as e.g. a DNA fragment with SEQ ID
NO:1 sequence or a DNA fragment containing the part or whole of the SEQ
ID NO:1 sequence to detect positive DNA fragment(s) from a test organism.
A DIG-labeled probe, e.g. SEQ ID NO:1, may be prepared according to
Example 1 by using the PCR-DIG labeling kit (Roche Diagnostics) and a set
of primers, SEQ ID NO:3 and SEQ ID NO:4 according to the supplier's
protocol. A result of such a blot is depicted in Table 1.

[0159]The hybridization may be performed under stringent or highly
stringent conditions. A preferred, non-limiting example of such
conditions are hybridization in 6× sodium chloride/sodium citrate
(SSC) at about 45° C., followed by one or more washes in
1×SSC, 0.1% SDS at 50° C., preferably at 55° C., more
preferably at 60° C. and even more preferably at 65° C.
Highly stringent conditions include, for example, 2 h to 4 days
incubation at 42° C. in a solution such as DigEasyHyb solution
(Roche Diagnostics GmbH) with or without 100 μg/ml salmon sperm DNA,
or a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM
trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1%
N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH),
followed by washing the filters twice for 5 to 15 min in 2×SSC and
0.1% SDS at room temperature and then washing twice for 15-30 min in
0.5×SSC and 0.1% SDS or 0.1×SSC and 0.1% SDS at 65-68°
C. To detect DNA fragments with lower identity to the probe DNA, final
washing steps can be done at lower temperatures such as 50-65° C.
and for shorter washing time such as 1-15 min.

[0160]The genes corresponding to the positive signals within the
respective organisms shown in Table 1 can be cloned by a PCR method well
known in the art using genomic DNA of such an organism together with a
suitable primer set, such as e.g. SEQ ID NO:3 and SEQ ID NO:4 under
conditions as described in Example 1 or as follows: 5 to 100 ng of
genomic DNA is used per reaction (total volume 50 μl). Expand High
Fidelity PCR system (Roche Diagnostics) can be used with reaction
conditions consisting of 94° C. for 2 min; 30 cycles of (i)
denaturation step at 94° C. for 15 sec, (ii) annealing step at
60° C. for 30 sec, (iii) synthesis step at 72° C. for 0.5
to 5 min depending to the target DNA length (1 min/1 kb); extension at
72° C. for 7 min. Alternatively, one can perform a PCR with
degenerate primers, which can be synthesized based on SEQ ID NO:2 or
amino acid sequences as consensus sequences selected by aligning several
amino acid sequences obtained by a sequence search program such as BLASTP
(or BLASTX when nucleotide sequence is used as a "query sequence") to
find proteins having a similarity to the protein of SEQ ID NO:2. For PCR
using degenerate primers, temperature of the second annealing step (see
above) can be lowered to 55° C., or even to 50-45° C. A
result of such an experiment is shown in Table 1.

[0161]Samples of the PCR reactions are separated by agarose gel
electrophoresis and the bands are visualized with a transilluminator
after staining with e.g. ethidium bromide, isolated from the gel and the
correct sequence is confirmed.

[0162]Consensus sequences mentioned above might be amino acid sequences
belonging to certain categories of several protein domain/family
databases such as PROSITE (database of protein families and domains),
COGs (Cluster of Ortholog Groups), CDD (Conserved Domain Databases), pfam
(large collection of multiple sequence alignments and hidden Markov
models covering many common protein domains and families). Once one can
select certain protein with identical/similar function to the protein of
this invention from proteins containing domain or family of such
databases, corresponding DNA encoding the protein can be amplified by PCR
using the protein sequence or its nucleotide sequence when it is
available in public databases.

Example 6

Disruption of the SMS 27 Gene and Equivalents in Other Organisms for
Production of Vitamin C and/or 2-KGA

[0164]The knockout mutant such as, e.g. a knockout mutant G. oxydans IFO
3292-SMS 27 equivalent gene::Km can be generated as follows: the PCR
product obtained from G. oxydans IFO 3293 described in Example 5 is
cloned in an E. coli vector pCR2.1-TOPO and used to transform E. coli TG1
to have a Apr transformant carrying pCR2.1-gene X. Then, Kmr
cassette isolated from pUC-4K (Amersham Bioscience, accession No. X06404)
is inserted into one of the restriction site of the target gene with
ligase and the resulting ligation product is used to transform E. coli
TG1 to have Apr Kmr transformant carrying pCR2.1-gene X::Km. The
pCR2.1-gene X::Km plasmid prepared from the transformant is digested by
two restriction enzymes selected from the multi-cloning site of the
vector part to isolate a DNA fragment containing gene X::Km. The
resulting DNA fragment is used to transform the host strain carrying the
SMS 27 equivalent gene by electroporation to have the gene disruptant
carrying SMS 27 equivalent gene::Km.

[0165]Further modifications including genes involved in the conversion of
D-sorbitol, L-sorbose and/or L-sorbosone into Vitamin C within said
strains may be generated to improve Vitamin C production within such
strains.

[0166]Production of Vitamin C using the cells of the knockout mutant, e.g.
G. oxydans IFO 3292-SMS 27 equivalent gene::Km, and the corresponding
wild-type strain, e.g. G. oxydans IFO 3292, are performed according to
Example 3.

[0167]Production of 2-KGA using the cells of the knockout mutant, e.g. G.
oxydans IFO 3292-SMS 27 equivalent gene::Km, and the corresponding
wild-type strain, e.g. G. oxydans IFO 3292, are performed according to
Example 4.

[0168]In the resting cell reaction with 1% L-sorbosone as the substrate,
the mutant strain can produce at least more than 20% Vitamin C and at
least more than 10% 2-KGA compared to the wild-type strain.